CN110241033B - Rhodotorula mucilaginosa JS2018 and application thereof in production of astaxanthin by fermenting molasses - Google Patents
Rhodotorula mucilaginosa JS2018 and application thereof in production of astaxanthin by fermenting molasses Download PDFInfo
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Abstract
The invention discloses rhodotorula mucilaginosa JS2018 and application thereof in production of astaxanthin by molasses fermentation. The Rhodotorula mucilaginosa JS2018 is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 5 and 30 months, and the preservation number is CGMCC No. 17871. The Rhodotorula mucilaginosa JS2018 with high astaxanthin yield is obtained by screening from soil, and is identified as Rhodotorula mucilaginosa (Rhodotorula muciniaosa) by morphological, metabolic and genetic identification. The strain can produce astaxanthin in a fermentation system containing molasses, sodium nitrate, ferrous sulfate and disodium hydrogen phosphate in high yield, optimizes the fermentation process, has the final biomass of 51.5g/L and the astaxanthin content of 4-6 percent of dry bacteria, can be used for industrial production of astaxanthin, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of microbial fermentation engineering, in particular to rhodotorula mucilaginosa JS2018 and application thereof in production of astaxanthin by molasses fermentation.
Background
Astaxanthin is a ketocarotenoid that is not a source of vitamin A. Animal experiments show that the astaxanthin has the functions of resisting tumor, ultraviolet radiation and immunity, and is a strong antioxidant and lipid peroxidation inhibitor for aquatic animals; it also has relieving effect on ophthalmic diseases, cardiovascular diseases and rheumatoid arthritis. It has been found that astaxanthin is useful for eliminating the discomfort caused by jet lag and is effective in comparison with conventional melatonin. In general, astaxanthin has application value in several aspects: the colorant is good for aquaculture and livestock breeding, and the survival rate, the weight gain rate and the feed conversion rate of the cultured products are improved; ② food additives (colorants and antioxidants); ③ health products and drugs (for enhancing immunity and preventing lipid peroxidation, etc.); fourthly, cosmetics (ultraviolet radiation resistance, oxidation resistance and the like).
Most of astaxanthin on the market is chemically synthesized, and a small amount of natural astaxanthin is also available. The chemically synthesized astaxanthin is mainly used as a fish feed additive, the industrial production of the chemically synthesized astaxanthin is controlled by Roche Switzerland, namely the current Tesmann company, the commercial name of the chemically synthesized astaxanthin is Carophylpink (Carophylpink), the astaxanthin content is 5-10%, but the food and health administration (FDA) does not allow any chemically synthesized new product to enter the health food market until now. Some animals and plants contain natural astaxanthin, although the extraction source is wide, the content is very low, such as about 80 μ g/g in waste shells of shrimps and crabs. Related studies have found that some microbial strains are capable of producing astaxanthin, such as Haematococcus Pluvialis and Phaffia rhodozyma. But there are not many microbial strains capable of producing astaxanthin in nature; in addition, some of the bacteria grow slowly and have low astaxanthin content, so that the method has no industrial application prospect. Although part of microorganisms are used for producing natural astaxanthin at present, the biomass and astaxanthin yield obtained by the prior fermentation technology are limited, and the production cost is high. Therefore, it is necessary to select a new microbial strain with high astaxanthin yield and develop a fermentation process thereof for industrial fermentation production of astaxanthin.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides Rhodotorula mucilaginosa JS 2018.
The invention also aims to provide application of the rhodotorula mucilaginosa JS2018 in production of astaxanthin by fermenting molasses.
The invention also aims to provide a method for producing astaxanthin by fermenting molasses through the rhodotorula mucilaginosa JS 2018.
In order to achieve the purpose, the invention is realized by the following scheme:
the Rhodotorula mucilaginosa JS2018 with high astaxanthin yield is obtained by screening from the nursery soil of the southern agricultural university of south China, the morphological, metabolic and genetic identification is carried out on the Rhodotorula mucilaginosa JS2018, and the 26S rDNA identification result verifies that the Rhodotorula mucilaginosa JS is Rhodotorula mucilaginosa. The strain can produce astaxanthin with high yield in a fermentation system containing molasses, sodium nitrate, ferrous sulfate and disodium hydrogen phosphate, a carbon source is supplemented according to growth conditions in the culture process, ventilation quantity and rotating speed are adjusted to control dissolved oxygen, the final biomass can reach 51.5g/L by optimizing the fermentation process, the astaxanthin content is 4% -6% of dry bacteria, and the strain can be used for industrial production of astaxanthin and has good application prospects.
Therefore, the Rhodotorula mucilaginosa strain JS2018 is claimed to be preserved in China general microbiological culture Collection center (CGMCC) in 2019, 5 and 30 months, and the preservation number is CGMCC No. 17871. The preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, the institute of microbiology of Chinese academy of sciences.
The rhodotorula mucilaginosa JS2018 disclosed by the invention has the following morphological, physiological and biochemical characteristics:
(1) morphological characteristics
The Rhodotorula mucilaginosa JS2018 is in a round or oval shape under a microscope and performs budding reproduction; on the PDA culture medium, the colony is round, pink orange, raised, neat in edge, smooth in surface and easy to pick up.
(2) Physiological and biochemical characteristics
The Rhodotorula mucilaginosa JS2018 is suitable for the growth temperature of 22-35 ℃. Rhodotorula mucilaginosa JS2018 can use glucose, fructose, maltose, galactose, sucrose, raffinose, xylose, melezitose, ribose, glycerol, trehalose and arabinose as carbon source for fermentation.
The 26S rDNA sequence of the rhodotorula mucilaginosa JS2018 is shown in SEQ ID NO: 1.
The invention also claims application of the rhodotorula mucilaginosa JS2018 in production of astaxanthin by fermenting molasses.
According to the invention, researches show that in the traditional batch culture, if the addition amount of an initial carbon source is not high enough, the obtained biomass and astaxanthin yield are low; however, if the addition amount of the initial carbon source is too high, the defects that cells grow in a large amount in the early stage, dissolved oxygen is insufficient, and a ventilation stirring device cannot adapt to the cells are caused, and the synthesis of astaxanthin is obviously inhibited, so that the final biomass and the yield of astaxanthin are not obviously improved, and the utilization rate of raw materials is low. The invention monitors the concentration of residual sugar, adopts a method of middle feeding to culture the rhodotorula mucilaginosa, avoids excessive feeding at one time, ensures higher total input amount, and adjusts the rotating speed and the ventilation volume according to the growth condition of thalli to ensure sufficient dissolved oxygen supply in the whole process, thereby obviously improving the biomass and the yield of astaxanthin.
Therefore, the invention claims a method for producing astaxanthin by fermenting molasses with the rhodotorula mucilaginosa JS2018, which comprises the following steps:
s1, adopting a large-capacity fermentation tank to inoculate rhodotorula mucilaginosa JS2018 into a fermentation culture medium for culture, wherein the fermentation culture medium contains molasses with total sugar content of 30g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O；
S2, when the concentration of residual sugar in the fermentation medium is lower than 10g/L, supplementing molasses for continuous culture;
the temperature of the whole fermentation period is controlled to be 26-32 ℃, the pH value is controlled to be 4.5-7.0, the rotating speed is 30-100 r/min, and the ventilation volume is 50-120 m3And h, dissolving oxygen by 40-55%, fermenting for 48-72 h, and separating and purifying to obtain astaxanthin.
Preferably, the inoculation amount of the rhodotorula mucilaginosa JS2018 in the step S1 is 3-8% (v/v).
More preferably, the Rhodotorula mucilaginosa JS2018 is inoculated in the amount of 6% (v/v) in step S1.
Preferably, the temperature is controlled at 30 ℃ and pH5.0 throughout the fermentation cycle.
Preferably, the whole fermentation period is controlled at a rotating speed of 60-80 r/min and an air flow of 80-100 m3H, dissolved oxygen 45%.
Preferably, the large capacity refers to the volume of a fermentation tank being 1000-5000L.
More preferably, the large capacity means that the volume of the fermenter is 5000L.
Compared with the prior art, the invention has the following beneficial effects:
the Rhodotorula mucilaginosa JS2018 with high astaxanthin yield is obtained by screening from soil, and is identified as Rhodotorula mucilaginosa (Rhodotorula muciniaosa) by morphological, metabolic and genetic identification. The strain can produce astaxanthin with high yield in a fermentation system containing molasses, sodium nitrate, ferrous sulfate and disodium hydrogen phosphate, a carbon source is supplemented according to growth conditions in the culture process, ventilation quantity and rotating speed are adjusted to control dissolved oxygen, the final biomass can reach 51.5g/L by optimizing the fermentation process, the astaxanthin content is 4% -6% of dry bacteria, and the strain can be used for industrial production of astaxanthin and has good application prospects.
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FIG. 1 shows the result of PCR amplification.
Fig. 2 is the morphological characteristics of rhodotorula mucilaginosa JS 2018. Wherein, a is the growth condition of rhodotorula mucilaginosa JS2018 on a PDA culture medium, and b is the shape of the rhodotorula mucilaginosa JS2018 under a microscope.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 screening and characterization of high astaxanthin-yielding Rhodotorula mucilaginosa
A new strain is separated from the soil of the nursery of the university of agriculture in south China, and the number of the new strain is JS 2018.
The strain was identified as Rhodotorula mucilaginosa (Rhodotorula mucor) by taxonomic identification using techniques such as 26S rDNA sequence analysis, as shown in FIG. 1 and Table 1.
TABLE 1 alignment of Gene sequences
Rhodotorula mucilaginosa JS2018 has the following morphological, physiological and biochemical characteristics:
(1) morphological characteristics
The Rhodotorula mucilaginosa JS2018 is in a round or oval shape under a microscope and performs budding reproduction; on PDA medium, the colony is round, pink orange, raised, neat in edge, smooth in surface, and easy to pick up (as shown in FIG. 2).
(2) Physiological and biochemical characteristics
The Rhodotorula mucilaginosa JS2018 is suitable for the growth temperature of 22-35 ℃. The strain can be fermented by using glucose, fructose, maltose, galactose, sucrose, raffinose, xylose, melezitose, ribose, glycerol, trehalose and arabinose as carbon sources. As shown in table 2.
TABLE 2 physiological and biochemical identification results (carbon source assimilation) of Strain JS2018
| Carbon source | Results | Carbon source | Results |
| Glucose | + | Fructose | + |
| Lactose | _ | Maltose | + |
| Galactose | + | Sucrose | + |
| Acetamide | _ | Rhamnose | _ |
| Mannitol | _ | Cotton seed candy | + |
| Cellobiose | _ | Melibiose | _ |
| Sorbitol | _ | Xylose | + |
| Melezitose | + | Ribose | + |
| Inositol | _ | Glycerol | + |
| Trehalose | + | Arab sugar | + |
The 26S rDNA sequence of the rhodotorula mucilaginosa JS2018 is shown in SEQ ID NO: 1.
The Rhodotorula mucilaginosa JS2018 is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 5 and 30 months, and the preservation number is CGMCC No. 17871. The preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, the institute of microbiology of Chinese academy of sciences.
Example 2
A method for producing astaxanthin by fermenting molasses with rhodotorula mucilaginosa JS2018 comprises the following steps:
s1, adopting 5000L fermentation tank middle feeding culture, inoculating rhodotorula mucilaginosa JS2018 into 3500L fermentation culture medium according to the inoculation amount of 6% (v/v), wherein the fermentation culture medium contains molasses with the total sugar amount of 30g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O；
S2, when the concentration of residual sugar in the fermentation medium is lower than 10g/L, supplementing molasses for continuous culture; the sugar supplementing mode is to control the flow rate to continuously supplement sugar, so that the sugar concentration is kept at about 15g/L until the total sugar amount reaches 120g/L (based on the volume of the initial culture medium);
the temperature of the whole fermentation period is controlled to be 30 ℃, the pH value is 5.0, the rotating speed is 60-80 r/min, and the ventilation volume is 80-100 m3And h, dissolving oxygen by 45%, fermenting for 72h, and separating and purifying to obtain astaxanthin.
The results show that the final biomass can reach 51.5g/L, and the astaxanthin content is 6% of dry thalli.
Example 3
A method for producing astaxanthin by fermenting molasses with rhodotorula mucilaginosa JS2018 comprises the following steps:
s1, adopting 5000L fermentation tank middle feeding culture, inoculating rhodotorula mucilaginosa JS2018 into 3500L fermentation culture medium according to the inoculation amount of 3% (v/v), wherein the fermentation culture medium contains molasses with the total sugar amount of 30g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O；
S2, when the concentration of residual sugar in the fermentation medium is lower than 10g/L, supplementing molasses for continuous culture; the sugar supplementing mode is to control the flow rate to continuously supplement sugar, so that the sugar concentration is kept at about 15g/L until the total sugar amount reaches 120g/L (based on the volume of the initial culture medium);
the temperature of the whole fermentation period is controlled to be 26 ℃, the pH value is 4.5, the rotating speed is 30-60 r/min, and the ventilation volume is 50-80 m3And h, dissolving oxygen by 40%, fermenting for 72h, and separating and purifying to obtain astaxanthin.
The results show that the final biomass can reach 42.5g/L, and the astaxanthin content is 5% of dry thalli.
Example 4
A method for producing astaxanthin by fermenting molasses with rhodotorula mucilaginosa JS2018 comprises the following steps:
s1, adopting 5000L fermentation tank middle feeding culture, inoculating rhodotorula mucilaginosa JS2018 into 3500L fermentation culture medium according to the inoculation amount of 8% (v/v), wherein the fermentation culture medium contains molasses with the total sugar amount of 30g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O；
S2, when the concentration of residual sugar in the fermentation medium is lower than 10g/L, supplementing molasses for continuous culture; the sugar supplementing mode is to control the flow rate to continuously supplement sugar, so that the sugar concentration is kept at about 15g/L until the total sugar amount reaches 120g/L (based on the volume of the initial culture medium);
the temperature of the whole fermentation period is controlled to be 32 ℃, the pH value is 6.0, the rotating speed is 80-100 r/min, and the ventilation volume is 100-120 m3And h, dissolving oxygen for 55%, fermenting for 72h, and separating and purifying to obtain astaxanthin.
The results show that the final biomass can reach 49.3g/L, and the astaxanthin content is 4% of dry thalli.
Example 5
A method for producing astaxanthin by fermenting molasses with rhodotorula mucilaginosa JS2018 comprises the following steps:
s1, adopting 5000L fermentation tank middle feeding culture, inoculating rhodotorula mucilaginosa JS2018 into 3500L fermentation culture medium according to the inoculation amount of 5% (v/v), wherein the fermentation culture medium contains molasses with the total sugar amount of 30g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O；
S2, when the concentration of residual sugar in the fermentation medium is lower than 10g/L, supplementing molasses for continuous culture; the sugar supplementing mode is to control the flow rate to continuously supplement sugar, so that the sugar concentration is kept at about 15g/L until the total sugar amount reaches 120g/L (based on the volume of the initial culture medium);
the temperature of the whole fermentation period is controlled to be 28 ℃, the pH value is 7.0, the rotating speed is 60-80 r/min, and the ventilation volume is 80-100 m3And h, dissolving oxygen by 50%, fermenting for 72h, and separating and purifying to obtain astaxanthin.
The results show that the final biomass can reach 45.6g/L, and the astaxanthin content is 5% of dry thalli.
Comparative example 1
A method for producing astaxanthin by fermenting molasses with rhodotorula mucilaginosa JS2018 comprises the following steps:
adopting a 5000L fermentation tank for batch culture (without intermediate feeding), inoculating rhodotorula mucilaginosa JS2018 into 3500L fermentation medium according to the inoculation amount of 6% (v/v), wherein the fermentation medium contains molasses with the total sugar amount of 120g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O; the temperature of the whole fermentation period is controlled to be 30 ℃, the pH value is 5.0, the rotating speed is 60-80 r/min, and the ventilation volume is 80-100 m3And h, dissolving oxygen by 45%, fermenting for 72h, and separating and purifying to obtain astaxanthin.
The results show that the final biomass can reach 28g/L, and the astaxanthin content is 2% of dry thalli.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> rhodotorula mucilaginosa JS2018 and application thereof in production of astaxanthin by molasses fermentation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 621
<212> DNA
<213> Rhodotorula mucilaginosa (Rhodotorula mucolignosa)
<400> 1
tccttccgta ggtgaacctg cggaaggatc attagtgaat ataggacgtc caacttaact 60
tggagtccga actctcactt tctaaccctg tgcacttgtt tgggatagta actctcgcaa 120
gagagcgaac tcctattcac ttataaacac aaagtctatg aatgtattaa attttataac 180
aaaataaaac tttcaacaac ggatctcttg gctctcgcat cgatgaagaa cgcagcgaaa 240
tgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcaccttg 300
cgctccatgg tattccgtgg agcatgcctg tttgagtgtc atgaatactt caaccctcct 360
ctttcttaat gattgaagag gtgtttggtt tctgagcgct gctggccttt acggtctagc 420
tcgttcgtaa tgcattagca tccgcaatcg aacttcggat tgacttggcg taatagacta 480
ttcgctgagg aattctagtc ttcggactag agccgggttg ggttaaagga agcttctaat 540
cagaatgtct acattttaag attagatctc aaatcaggta ggactacccg ctgaacttaa 600
gcatatcaat aaggcgggag g 621
Claims (7)
1. A Rhodotorula mucilaginosa strain JS2018 is characterized in that the Rhodotorula mucilaginosa strain JS2018 is preserved in China general microbiological culture Collection center in 2019, 5 and 30 months, and the preservation number is CGMCC No. 17871.
2. The use of the rhodotorula mucilaginosa JS2018 as claimed in claim 1 for the production of astaxanthin by fermenting molasses.
3. A method for producing astaxanthin by fermenting molasses with rhodotorula mucilaginosa JS2018 as claimed in claim 1, comprising the steps of:
s1, adopting a 1000-5000L fermentation tank, inoculating rhodotorula mucilaginosa JS2018 into a fermentation culture medium according to the inoculation amount of 3-8% of the volume ratio for culture, wherein the fermentation culture medium contains molasses with the total sugar amount of 30g/L, 10g/L sodium nitrate and 0.1g/L FeSO4·7H2O、4.5g/L Na2HPO4·12H2O；
S2, when the concentration of residual sugar in the fermentation medium is lower than 10g/L, supplementing molasses for continuous culture; the temperature of the whole fermentation period is controlled to be 26-32 ℃, the pH value is controlled to be 4.5-7.0, the rotating speed is 30-100 r/min, and the ventilation volume is 50-120 m3And h, dissolving oxygen by 40-55%, fermenting for 48-72 h, and separating and purifying to obtain astaxanthin.
4. The method according to claim 3, wherein the Rhodotorula mucilaginosa JS2018 is inoculated in an amount of 6% by volume in step S1.
5. The method according to claim 3, wherein the temperature is controlled at 30 ℃ and pH5.0 throughout the fermentation cycle.
6. The method according to claim 3, wherein the whole fermentation period is controlled at a rotation speed of 60-80 r/min and an air flow of 80-100 m3H, dissolved oxygen 45%.
7. The method of claim 3, wherein the volume of the fermentor is 5000L.
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